Methods of altering an immune response induced by CpG oligodeoxynucleotides

ABSTRACT

It is disclosed herein that agents that affect the activity and/or expression of CXCL16 can be used to alter the uptake of D-type CpG oligodeoxynucleotides (D ODNs). Methods of inducing an immune response are disclosed that include administering agents that increase the activity and/or expression of CXCL16 and a D ODN. Methods of decreasing an immune response to a CpG ODN are also disclosed. These methods include administering an agent that decreases the activity and/or expression of CXCL16. Compositions including one or more D-type ODNs and an agent that modulates that activity and/or expression of CXCL16 are provided.

PRIORITY CLAIM, CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.12/065,085, filed Feb. 27, 2008 now U.S. Pat. No. 7,892,569, which isthe U.S. National Stage of International Application No.PCT/US2006/033774, filed Aug. 28, 2006, which was published in Englishunder PCT Article 21(2), which in turn claims the benefit of U.S.Provisional Application No. 60/713,547, filed Aug. 31, 2005. The priorapplications are incorporated herein by reference in their entirety.

FIELD

This application relates to the field of immunology, specifically toagents that can be used to alter the uptake of immunostimulatoryoligodeoxynucleotides (ODNs).

BACKGROUND

DNA is a complex macromolecule whose activities are influenced by itsbase composition and base modification, as well as helical orientation.Bacterial DNA, as well as certain synthetic oligodeoxynucleotides (ODNs)containing unmethylated CpG sequences, can induce proliferation andimmunoglobulin production by murine B cells. Unmethylated CpGdinucleotides are more frequent in the genomes of bacteria and virusesthan vertebrates. Studies have suggested that immune recognition ofthese motifs may contribute to the host's innate immune response.(Klinman et al., Proc. Natl. Acad. Sci. USA 93:2879, 1996; Yi et al., J.Immun. 157:5394, 1996; Liang et al., J. Clin. Invest. II 9:89, 1996;Krieg et al., Nature 374:546, 1995).

A CpG oligodeoxynucleotide (ODN) is an oligodeoxynucleotide including aCpG motif, wherein the pyrimidine ring of the cytosine is unmethylated.Three types of CpG ODNs have been identified: C-type, K-type and D-typeODNs. Generally, CpG ODNs range from about 8 to 30 bases in size. D- andK-type nucleic acid sequences have been described in the published PCTPublication No. WO 98/18810A1 (K-type) and published PCT Publication No.WO 00/61151 (D-type). Generally D ODNs can stimulate a cellular immuneresponse, while K ODNs can stimulate a humoral immune response.

Unmethylated CpG motifs, including both D-type ODNs and K-type ODNs, arerecognized by the Toll-like receptor 9 (TLR9) expressed on immune cells(such as B cells, macrophages, and dendritic cells). The CpG DNA istaken up by an endocytic/phagocytic pathway. It is known that theinteraction of CpG ODN with TLR9 triggers recruitment of a MyD88 adaptormolecule, activation of an IL-1R kinase-1 and other factors, resultingin the production of cytokines (see Latz et al., Nat. Immunol. 5:190-8,2004).

CpG ODNs can be used to induce an immune response. Thus, they have beenfound to have many uses, such as to induce an immune response toantigens, in the production of vaccines, and as adjuvants. It would beadvantageous to be able to alter the uptake of CpG ODN by cells, inorder to alter the immune response produced by these oligonucleotides.Methods to alter the uptake and subsequent immune activation triggeredby CpG ODN are disclosed herein.

SUMMARY

It is disclosed herein that agents that affect the activity and/orexpression of CXCL16 can be used to alter the uptake of CpGoligodeoxynucleotides (ODN), specifically D-type CpGoligodeoxynucleotides (D ODN). Thus, agents that affect the activityand/or expression of CXCL16 can be used to alter an immune responseinduced by D ODN. In one example, the agent increases the activityand/or expression of CXCL16, thereby increasing the uptake of D ODN.Agents that increase the activity and/or expression of CXCL16 can beused to increase an immune response induced by a D ODN. Agents thatdecrease the activity and/or expression of CXCL16 can be used todecrease an immune response induced by D ODN.

Specific compositions including one or more D-type ODNs and an agentthat modulates that activity and/or expression of CXCL16 are providedherein. These compositions are of use to induce an immune response, suchas to a specific antigen.

The foregoing and other features and advantages will become moreapparent from the following detailed description of several embodiments,which proceeds with reference to the accompanying figures.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A-1C are graphs and digital images illustrating that CXCL16selectively recognizes D ODN. FIG. 1A is a line graph showing CpG ODNbinding to recombinant CXCL16. 96-well flat bottom ELISA plates werecoated with 0.4 μg/ml anti-CXCL16 antibody and then incubated without(dotted lines) or with 200 ng/ml of recombinant CXCL16 (full lines).Following washing, 1, 0.2 or 0.04 μM biotin-conjugated K or Doligodeoxynucleotide (ODN) were added. After washing, ODN binding wasdetected colorimetrically using phosphatase-conjugated avidin followedby a phosphatase specific colorimetric substrate. Results are presentedas average±standard deviation (SD) of three independent readings. FIG.1B is a set of digital images and plots showing that D but not K ODNcolocalize with CXCL16 and its uptake is enhanced in transfected HEK293cells. CXCL16 transfected HEK293 cells were incubated with 3 μM of FITCconjugated CpG ODN at 37° C. for 20 minutes. Cells were stained forCXCL16 expression (left) and colocalization (bright cells, right) withODN (middle) was determined using confocal microscopy. Percent ofFITC-ODN bright cells in mock transfected (filled histograms) and CXCL16transfected (dark open histograms) was determined using flow cytometryand are shown against background staining (dotted histograms). Resultsare representative of at least six independent experiments. FIG. 1C is abar graph showing that anti-CXCL16 inhibit D ODN binding to pDC. Bindingof FITC conjugated ODN to CD123/BDCA-2 double positive pDC wasdetermined in the absence or presence of anti-CXCL16 or isotype matchedcontrol (100 μg/ml each). Percent inhibition of uptake was determinedcomparative to the isotype matched control group. Results represent themean±SD of 2 experiments (*, P<0.05; **, P<0.01).

FIGS. 2A-2D are graphs and plots showing that CXCL16 positive cellspreferentially respond to D ODN. FIG. 2A is a plot showing that CXCL16expression versus cytokine production in pDCs stimulated with CpG ODN.Peripheral blood mononuclear cells (PBMC) (4×10⁶/ml) were stimulatedwith Control, K or D ODN in the presence of Brefeldin A (10 μg/ml) for4.5 h (TNF-α) or for 12 hours (Brefeldin A was added after 8 hours ofincubation) in the case of IFN-α. Cytokine producing cells were assessedfrom the CD123⁺-gated cells as a function of CXCL16 expression. Resultsare representative of 3 independent experiments. FIG. 2B is a bar graphshowing that antibodies against CXCL16 inhibit D ODN induced cytokineproduction. PBMC were preincubated with 25 μg/ml of isotype (open bars)or anti-CXCL16 antibody (filled bars) for 30 minutes at 37° C. and thenstimulated with 1 μM each of K or with 3 μM D ODN for 24 hours. Percentinhibition of cytokine production was calculated from three differentindividuals (*, P<0.05). FIG. 2C is a plot showing the effect ofanti-CXCL16 on the upregulation of HLA-DR and CD86 by CpG ODN.Elutriated monocytes were preincubated with anti-CXCL16 or isotypematched control antibody and then stimulated with Control (3 μM), K (1μM) or D (3 μM) ODN for 24 hours. The percent of cells expressing HLA-DRand CD86 was determined by flow cytometry. Results are representative offive independent experiments. FIG. 2D is a bar graph of the resultsshowing HEK293 cells transfected with CXCL16 gain responsiveness to DODN. HEK293 cells stably expressing TLR9 were co-transfected withp5×NF-kB. In this figure, the ratio of NF-kB induction in CXCL16transfected over mock transfected is displayed for each ODN. All ODNnames are shown on the X-axis. B-luciferase plus control plasmid orCXCL16. Relative luciferase units from CXCL16 transfected cells overthose from mock transfected cells were determined 24 hours after ODNtreatment (3 μM). Results represent the mean±SD of 4 independentexperiments (*, P<0.05; **, P<0.01).

FIGS. 3A-3D are bar graphs and plots showing the effect of scavengerreceptor ligands on the binding and activity of CpG ODN. FIG. 3A is abar graph of the results obtained when purified pDC were preincubated(20 minutes at 4° C.) with medium or with 50 μg/ml each of inhibitorsfollowed by addition of 1 μM FITC conjugated ODN. Mean fluorescenceintensity representing binding of FITC-ODN was assessed using flowcytometry. Results represent the mean±S.D of three independentexperiments. FIG. 3B is a bar graph showing D ODN induced IFN-α fromperipheral blood mononuclear cells (PBMC). Open bars: medium; dottedbars: chondroitin sulfate; right-striped bars: fucoidan; left-stripedbars: dextran sulfate Production of IFN-α from PBMC (in the absence orpresence of 50 μg/ml inhibitors) was determined by ELISA from 24 hourculture supernatants. FIG. 3C is a plot showing K ODN induced TNF-αproduction in pDC (in the absence or presence of 50 μg/ml dextransulfate). TNF-α production was determined using intracytoplasmiccytokine staining (representative plots of three independentexperiments). FIG. 3D is a plot showing the results obtained whenpurified pDC were incubated with 1 μM each of K or with 3 μM D ODN inthe absence or presence of 50 μg/ml dextran sulfate. Upregulation ofHLA-DR/CD54 was determined 24 hours later using flow cytometry.

FIGS. 4A-4B are plots showing the expression of CXCL16. FIG. 4A showspDCs express CXCL16 on their surface. pDCs were enriched from PBMC usingthe BDCA-4 magnetic cell separation kit. Expression of CXCL16 versusisotype matched control was analyzed on CD123/BDCA-2 double positivecells. Data is representative of at least five independent experiments.FIG. 4B is a plot showing that anti-CXCL16 specifically inhibits bindingof D ODN to pDC. Binding of FITC conjugated D ODN to CD123 gated cellswas analyzed using flow cytometry. The figure shows that both basophils(BDCA-2^(negative)) and pDCs (BDCA-2^(positive)) bind D ODN in thepresence of isotype matched antibody and that only binding to the pDCbut not to the basophil population (which do not express CXCL16) isinhibited by anti-CXCL16, providing evidence for the specificity of thisantibody.

FIG. 5 is a set of plots of the results obtained when elutriatedmonocytes were stained for pDC markers and CXCL16 and BDCA-2/CD123 gatedcells were sorted into the CXCL16 negative or CXCL16 positive+negativepopulations using FACSAria cell sorter. The resulting populations werethen incubated with 1 and 3 μM of K and D ODN, respectively, or with1×107 pfu/ml of UV-inactivated HSV-1. Twenty-four hours later, cellswere stained and expression of HLA-DR/CD86 was assessed by flowcytometry.

FIG. 6 is a bar graph showing the expression of CXCL16 in HEK293 cellsstably expressing TLR9 confers D ODN responsiveness. Stable TLR9expressing HEK293 cells were co-transfected with 0.5 μg p5×NF-kB B-lucand 1 μg of CXCL16 or control plasmid for 24 hours. Data show relativeluciferase units obtained following 24 hours incubation with 3 μM ODN inmock transfected (open bars) and CXCL16 (filled bars) transfected TLR9expressing HEK293 cells.

FIGS. 7A-7B are a plot and a bar graph showing the metalloproteinaseinhibitor GM-6001 enhances CXCL16 expression and subsequent cytokineproduction induced by D ODN. PBMC were preincubated in the absence (openbars) or presence (filled bars) of GM-6001 (50 μM) for 30 minutes,washed and then stained for CXCL16 expression on pDC (see FIG. 7A) orstimulated with 1 μM of K or 3 μM D ODN for 24 hours (FIG. 7B). Cytokineproduction (IL-6 for K ODN and IFN-α D ODN) was assessed from culturesupernatants using ELISA. Individual results from six different PBMC areshown.

SEQUENCE LISTING

The nucleic and amino acid sequences listed in the accompanying sequencelisting are shown using standard letter abbreviations for nucleotidebases, and three letter code for amino acids, as defined in 37 C.F.R.1.822. Only one strand of each nucleic acid sequence is shown, but thecomplementary strand is understood as included by any reference to thedisplayed strand where appropriate.

The Sequence Listing is submitted as an ASCII text file[Sequence_Listing_(—)4239-70295-08.txt, Jan. 12, 2011, 6.13 KB], whichis incorporated by reference herein.

SEQ ID NOs: 1-17 are the nucleic acid sequences of D ODN.

SEQ ID NOs: 18-20 are the nucleic acid sequences of CXCL16 probes andprimers.

SEQ ID NO: 21 is the consensus nucleic acid sequence of a D ODN.

SEQ ID NO: 22 is the consensus nucleic aid sequence of a K ODN.

The Sequence Listing is submitted as an ASCII text file

DETAILED DESCRIPTION

I. Terms

Unless otherwise noted, technical terms are used according toconventional usage. Definitions of common terms in molecular biology maybe found in Benjamin Lewin, Genes V, published by Oxford UniversityPress, 1994 (ISBN 0-19-854287-9); Kendrew et al. (eds.), TheEncyclopedia of Molecular Biology, published by Blackwell Science Ltd.,1994 (ISBN 0-632-02182-9); and Robert A. Meyers (ed.), Molecular Biologyand Biotechnology: a Comprehensive Desk Reference, published by VCHPublishers, Inc., 1995 (ISBN 1-56081-569-8).

In order to facilitate review of the various embodiments of thisdisclosure, the following explanations of specific terms are provided:

ADAM-10: A protein that is an enzyme also known as disintegrin,metalloprotease domain 10, and MADM mammalian disintegrinmetalloprotease. It is a member of the ADAM protein family. The proteinhas been shown to be a physiologically relevant TNF-processing enzyme(TNF-alpha convertase, TNF-alpha converting enzyme). It cleaves the 26kDa membrane-bound precursor form of TNF-alpha to release the solublemature 17 kDa TNF form.

ADAM-10 contains the canonical zinc metalloproteinase motif, and hasbeen shown to be proteolytically active. ADAM-10 from bovine kidney wasshown to have type-IV collagenolytic activity, making ADAM-10 a“gelatinase.” ADAM-10 is efficiently inhibited by the endogenous MMPinhibitors TIMP-1 and TIMP-3, but not by TIMP-2 and TIMP-4.

The full length ADAM-10 sequence codes for a 748 amino acid protein,with a predicted mass, is 84.142 kD. Glycosylation and the cyteine-richregions make the protein run at 98 kD on reduced SDS PAGE, and 60-58 kDwhen Furin processed. A smaller 691 amino acid sequence for ADAM-10,lacking the transmembrane domain, has been reported, with a predictedmolecular weight of 77.633 kD. ADAM-10 is thought to bemembrane-anchored under normal conditions. The sequence of ADAM-10 canbe found, for example, as GENBANK™ Accession Nos. AAC51766 (Sep. 26,1997), AF009615 (Sep. 27, 1997) and CAA88463 (Apr. 18, 2005, MADAM),which are all incorporated herein by reference.

Animal: Living multi-cellular vertebrate organisms, a category thatincludes, for example, mammals and birds. The term mammal includes bothhuman and non-human mammals. Similarly, the term “subject” includes bothhuman and veterinary subjects.

Antigen: A compound, composition, or substance that can stimulate theproduction of antibodies or a T cell response in an animal, includingcompositions that are injected or absorbed into an animal. An antigenreacts with the products of specific humoral or cellular immunity,including those induced by heterologous immunogens. The term “antigen”includes all related antigenic epitopes.

“C” Class oligodeoxynucleotides (ODNs): ODNs that resemble K ODNs andare composed of only phosphorothiote nucleotides. Typically, C classODNs have a TCGTCG motif at the 5′ end and have a CpG motif imbedded ina palindromic sequence. Backbone modifications like 2′-O-methylmodifications especially in the 5′ part of the ODN influenceIFN-alpha-producing capacity of these ODN. C class ODNs have combinedproperties of D- and K-type ODNs. This class of ODNs stimulates B cellsto secrete IL-6 and stimulates plasmacytoid dendritic cells to produceinterferon-α. C class ODNs also induce IP-10 production and strong NKactivation.

CpG or CpG Motif: A nucleic acid having a cytosine followed by a guaninelinked by a phosphate bond in which the pyrimidine ring of the cytosineis unmethylated. The term “methylated CpG” refers to the methylation ofthe cytosine on the pyrimidine ring, usually occurring at the 5-positionof the pyrimidine ring. A CpG motif is a pattern of bases that includean unmethylated central CpG surrounded by at least one base flanking (onthe 3′ and the 5′ side of) the central CpG. Without being bound bytheory, the bases flanking the CpG confer part of the activity to theCpG oligodeoxynucleotide. A CpG oligonucleotide is an oligonucleotidethat is at least about ten nucleotides in length and includes anunmethylated CpG. CpG oligonucleotides include both D- and K-typeoligodeoxynucleotides (see below). CpG oligodeoxynucleotides aresingle-stranded. The entire CpG oligodeoxynucleotide can be unmethylatedor portions may be unmethylated. In one embodiment, at least the C ofthe 5′ CG 3′ is unmethylated.

Cancer: A malignant neoplasm that has undergone characteristic anaplasiawith loss of differentiation, increased rate of growth, invasion ofsurrounding tissue, and is capable of metastasis. For example, thyroidcancer is a malignant neoplasm that arises in or from thyroid tissue,and breast cancer is a malignant neoplasm that arises in or from breasttissue (such as a ductal carcinoma). Residual cancer is cancer thatremains in a subject after any form of treatment given to the subject toreduce or eradicate the cancer. Metastatic cancer is a cancer at one ormore sites in the body other than the site of origin of the original(primary) cancer from which the metastatic cancer is derived.

Chemotherapy or Chemotherapeutic Agents: As used herein, any chemicalagent with therapeutic usefulness in the treatment of diseasescharacterized by abnormal cell growth. Such diseases include tumors,neoplasms, and cancer as well as diseases characterized by hyperplasticgrowth such as psoriasis. In one embodiment, a chemotherapeutic agent isan agent of use in treating neoplasms such as solid tumors. In oneembodiment, a chemotherapeutic agent is a radioactive molecule. One ofskill in the art can readily identify a chemotherapeutic agent of use(e.g. see Slapak and Kufe, Principles of Cancer Therapy, Chapter 86 inHarrison's Principles of Internal Medicine, 14th edition; Perry et al.,Chemotherapy, Ch. 17 in Abeloff, Clinical Oncology 2^(nd) ed., © 2000Churchill Livingstone, Inc; Baltzer L, Berkery R (eds): Oncology PocketGuide to Chemotherapy, 2nd ed. St. Louis, Mosby-Year Book, 1995; FischerD S, Knobf M F, Durivage H J (eds): The Cancer Chemotherapy Handbook,4th ed. St. Louis, Mosby-Year Book, 1993). Chemotherapeutic agentsinclude those known by those skilled in the art, including but notlimited to: 5-fluorouracil (5-FU), azathioprine, cyclophosphamide,antimetabolites (such as Fludarabine), antineoplastics (such asEtoposide, Doxorubicin, methotrexate, and Vincristine), carboplatin,cis-platinum and the taxanes, such as taxol. Rapamycin has also beenused as a chemotherapeutic.

CXCL16: A chemokine that specifically binds to the CXCL16 receptor(CXCR6, also known as Bonzo), also known as SR-PSOX. Exemplary aminoacid sequences for CXCL16, and nucleotide sequence(s) encoding CXCL16are set forth as GENBANK™/EMBL Data Bank as Accession Nos. AF275260(human SR-PSOX, Jan. 2, 2001), AF277001 (murine SR-PSOX, Jan. 8, 2001),and AF277000 (porcine SR-PSOX, Jan. 8, 2001), which are all incorporatedherein by reference). Variants of CXCL16 that bind to their receptor arealso encompassed by this disclosure. The CXCL16 receptor is a type Imembrane protein which is expressed on macrophages and dendritic cellsand has a molecular weight of approximately 30 KDa.

CXCL16 is a ligand for the CXC-chemokine receptor CXCR6, and is ascavenger receptor for oxidized low density lipoprotein (LDL). CXCL16 isexpressed on the cell membrane as a multidomain molecule including achemokine domain followed by a glycosylated mucin-like stalk and singletransmembrane helix followed by a short cytoplasmic tail. CXCL16 isexpressed on antigen presenting cells (APCs). CXCL16 induces chemotaxisof activated T cells and bone marrow plasma cells. Cell expressed CXCL16is released from the cell membrane by proteolytic cleavage. Thedisintegrin-like metalloproteinase ADAM-10 plays a role in CXCL16cleavage. CXCL16 is induced by IFN-γ and TNF-α.

Cytokine: Proteins made by cells that affect the behavior of othercells, such as lymphocytes. In one embodiment, a cytokine is achemokine, a molecule that affects cellular trafficking.

D-Type Oligodeoxynucleotide (D ODN): An oligodeoxynucleotide includingan unmethylated CpG motif that has a sequence represented by theformula:

5′ RY-CpG-RY 3′wherein the central CpG motif is unmethylated, R is A or G (a purine),and Y is C or T (a pyrimidine). D-type oligodeoxynucleotides include anunmethylated CpG dinucleotide. Inversion, replacement or methylation ofthe CpG reduces or abrogates the activity of the D oligodeoxynucleotide.

In one embodiment, a D-type ODN is at least about 16 nucleotides inlength and includes a sequence represented by the formula:

(SEQ ID NO: 21) 5′X₁X₂X₃ Pu₁ Py₂ CpG Pu₃ Py₄ X₄X_(S)X₆(W)_(M) (G)_(N)-3′wherein the central CpG motif is unmethylated, Pu is a purinenucleotide, Py is a pyrimidine nucleotide, X and W are any nucleotide, Mis any integer from 0 to 10, and N is any integer from 4 to 10. Anadditional detailed description of D ODN sequences and their activitiescan be found in Verthelyi et al., J. Immunol. 166:2372-2377, 2001, whichis herein incorporated by reference. Generally D ODNs can stimulate acellular response. For example, an “effective amount” or“therapeutically effective amount” of a D ODN is an amount of the D ODNsufficient to stimulate a response.

Epitope: An antigenic determinant. These are particular chemical groupsor peptide sequences on a molecule that are antigenic, i.e. that elicita specific immune response. An antibody binds a particular antigenicepitope.

Functionally Equivalent: Sequence alterations, for example in a D-typeODN, that yield the same results as described herein. Such sequencealterations can include, but are not limited to, deletions, basemodifications, mutations, labeling, and insertions.

Immune Response: A response of a cell of the immune system, such as a Bcell, or a T cell, to a stimulus. In one embodiment, the response isspecific for a particular antigen (an “antigen-specific response”).

A “parameter of an immune response” is any particular measurable aspectof an immune response, including, but not limited to, cytokine secretion(IL-6, IL-10, IFN-α, etc.), immunoglobulin production, dendritic cellmaturation, and proliferation of a cell of the immune system. One ofskill in the art can readily determine an increase in any one of theseparameters, using known laboratory assays. In one specific non-limitingexample, to assess cell proliferation, incorporation of ³H-thymidine canbe assessed. A “substantial” increase in a parameter of the immuneresponse is a significant increase in this parameter as compared to acontrol. Specific, non-limiting examples of a substantial increase areat least about a 50% increase, at least about a 75% increase, at leastabout a 90% increase, at least about a 100% increase, at least about a200% increase, at least about a 300% increase, and at least about a 500%increase. One of skill in the art can readily identify a significantincrease using known statistical methods. One specific, non-limitingexample of a statistical test used to assess a substantial increase isthe use of a Z test to compare the percent of samples that respond to aD ODN as compared to the percent of samples that respond to a control. Anon-paramentric ANOVA can be used to compare differences in themagnitude of the response induced by D ODN as compared to the percent ofsamples that respond using a control. In this example, p<0.05 issignificant, and indicates a substantial increase in the parameter ofthe immune response. One of skill in the art can readily identify otherstatistical assays of use.

Infectious Agent: An agent that can infect a subject, including, but notlimited to, viruses, bacteria, and fungi.

Examples of infectious virus include: Retroviridae; Picornaviridae (forexample, polio viruses, hepatitis A virus; enteroviruses, humancoxsackie viruses, rhinoviruses, echoviruses); Calciviridae (such asstrains that cause gastroenteritis); Togaviridae (for example, equineencephalitis viruses, rubella viruses); Flaviridae (for example, dengueviruses, encephalitis viruses, yellow fever viruses); Coronaviridae (forexample, coronaviruses); Rhabdoviridae (for example, vesicularstomatitis viruses, rabies viruses); Filoviridae (for example, ebolaviruses); Paramyxoviridae (for example, parainfluenza viruses, mumpsvirus, measles virus, respiratory syncytial virus); Orthomyxoviridae(for example, influenza viruses); Bungaviridae (for example, Hantaanviruses, bunga viruses, phleboviruses and Nairo viruses); Arena viridae(hemorrhagic fever viruses); Reoviridae (e.g., reoviruses, orbiviursesand rotaviruses); Birnaviridae; Hepadnaviridae (Hepatitis B virus);Parvoviridae (parvoviruses); Papovaviridae (papilloma viruses, polyomaviruses); Adenoviridae (most adenoviruses); Herpesviridae (herpessimplex virus (HSV) 1 and HSV-2, varicella zoster virus, cytomegalovirus(CMV), herpes viruses); Poxviridae (variola viruses, vaccinia viruses,pox viruses); and Iridoviridae (such as African swine fever virus); andunclassified viruses (for example, the etiological agents of Spongiformencephalopathies, the agent of delta hepatitis (thought to be adefective satellite of Hepatitis B virus), the agents of non-A, non-BHepatitis (class 1=internally transmitted; class 2=parenterallytransmitted (i.e., Hepatitis C); Norwalk and related viruses, andastroviruses).

Examples of infectious bacteria include: Helicobacter pyloris; Boreliaburgdorferi; Legionella pneumophilia; Mycobacteria sps (such as, M.tuberculosis, M. avium, M. intracellulare, M. kansaii, M. gordonae);Staphylococcus aureus, Neisseria gonorrhoeae; Neisseria meningitidis;Listeria monocytogenes; Streptococcus pyogenes (Group A Streptococcus);Streptococcus agalactiae (Group B Streptococcus); Streptococcus(viridans group); Streptococcus faecalis; Streptococcus Bovis;Streptococcus (anaerobic sps); Streptococcus pneumoniae; pathogenicCampylobacter sp.; Enterococcus sp.; Haemophilus influenzae; Bacillusantracis; corynebacterium diphtheriae; corynebacterium sp.;Erysipelothrix rhusiopathiae; Clostridium perfringers; Clostridiumtetani; Enterobacter aerogenes; Klebsiella pneumoniae; Pasturellamultocida; Bacteroides sp.; Fusobacterium nucleatum; Streptobacillusmoniliformis; Treponema pallidum; Treponema pertenue; Leptospira; andActinomyces israelli.

Examples of infectious fungi include, but are not limited to,Cryptococcus neoformans; Histoplasma capsulatum; Coccidioides immitis;Blastomyces dermatitidis; Chlamydia trachomatis; and Candida albicans.

Other infectious organisms (such as protists) include: Plasmodiumfalciparum and Toxoplasma gondii.

Interferon alpha (α): IFN-α forms are produced by monocytes/macrophages,lymphoblastoid cells, fibroblasts, and a number of different cell typesfollowing induction by viruses, nucleic acids, glucocorticoid hormones,and low-molecular weight substances (n-butyrate, 5-bromodeoxy uridine).At least 23 different variants of IFN-α are known. The individualproteins have molecular masses between 19-26 kDa and consist of proteinswith lengths of 156-166 and 172 amino acids.

All IFN-α subtypes possess a common conserved sequence region betweenamino acid positions 115-151 while the amino-terminal ends are variable.Many IFN-α subtypes differ in their sequences at only one or twopositions. Naturally occurring variants also include proteins truncatedby 10 amino acids at the carboxy-terminal end. Disulfide bonds areformed between cysteines at positions 1/98 and 29/138. The disulfidebond 29/138 is essential for biological activity while the 1/98 bond canbe reduced without affecting biological activity.

There are at least 23 different IFN-α genes. They have a length of 1-2kb and are clustered on human chromosome 9p22. IFN-α genes do notcontain intron sequences found in many other eukaryotic genes. Basedupon the structures two types of IFN-α genes, designated class I and II,are distinguished. They encode proteins of 156-166 and 172 amino acids,respectively.

All known subtypes of IFN-α show the same antiviral antiparasitic,antiproliferative activities in suitable bioassays although they maydiffer in relative activities. Human IFN-α is also a potent antiviralsubstance in murine, porcine, and bovine cell systems. A number ofassays for IFN-α have been described. For example, IFN-α can be assayedby a cytopathic effect reduction test employing human and bovine celllines. Minute amounts of IFN-α can be assayed also by detection of theMx protein specifically induced by this interferon. A sandwich ELISAemploying bispecific monoclonal antibodies for rapid detection (10units/mL=0.1 ng/mL within 2-3 hours) is also available.

Interferon Gamma (γ): IFN-γ is a dimeric protein with subunits of 146amino acids. The protein is glycosylated at two sites, and the pI is8.3-8.5. IFN-γ is synthesized as a precursor protein of 166 amino acidsincluding a secretory signal sequence of 23 amino acids. Two molecularforms of the biologically active protein of 20 and 25 kDa have beendescribed. Both of them are glycosylated at position 25. The 25 kDa formis also glycosylated at position 97. The observed differences of naturalIFN-γ with respect to molecular mass and charge are due to variableglycosylation patterns. 40-60 kDa forms observed under non-denaturingconditions are dimers and tetramers of IFN-γ. The human gene has alength of approximately 6 kb. It contains four exons and maps tochromosome 12q24.1.

IFN-γ can be detected by sensitive immunoassays, such as an ELSA testthat allows detection of individual cells producing IFN-γ. Minuteamounts of IFN-γ can be detected indirectly by measuring IFN-inducedproteins such as Mx protein. The induction of the synthesis of IP-10 hasbeen used also to measure IFN-γ concentrations. In addition, bioassayscan be used to detect IFN-γ, such as an assay that employs induction ofindoleamine 2,3-dioxygenase activity in 2D9 cells.

Isolated: An “isolated” biological component (such as a nucleic acid,peptide or protein) has been substantially separated, produced apartfrom, or purified away from other biological components in the cell ofthe organism in which the component naturally occurs, i.e., otherchromosomal and extrachromosomal DNA and RNA, and proteins. Nucleicacids, peptides and proteins which have been “isolated” thus includenucleic acids and proteins purified by standard purification methods.The term also embraces nucleic acids, peptides and proteins prepared byrecombinant expression in a host cell as well as chemically synthesizednucleic acids.

K-Type Oligodeoxynucleotide (K ODN): An oligodeoxynucleotide includingan unmethylated CpG motif that has a sequence represented by theformula:

5′ N₁N₂N₃Q-CpG-WN₄N₅N₆ 3′ (SEQ ID NO: 22)

wherein the central CpG motif is unmethylated, Q is T, G or A, W is A orT, and N₁, N₂, N₃, N₄, N₅, and N₆ are any nucleotides. In oneembodiment, Q is a T. An additional detailed description of K ODNsequences and their activities can be found below. Generally K ODNs canstimulate a humoral response. For example, K ODNs stimulate theproduction of immunoglobulins, such as IgM and IgG. K ODNs can alsostimulate proliferation of peripheral blood mononuclear cells andincrease expression of IL-6 and/or IL-12, amongst other activities.

Leukocyte: Cells in the blood, also termed “white cells,” that areinvolved in defending the body against infective organisms and foreignsubstances. Leukocytes are produced in the bone marrow. There are 5 maintypes of white blood cells, subdivided between 2 main groups:polymorphonuclear leukocytes (neutrophils, eosinophils, basophils) andmononuclear leukocytes (monocytes and lymphocytes). When an infection ispresent, the production of leukocytes increases.

Mammal: This term includes both human and non-human mammals. Similarly,the term “subject” includes both human and veterinary subjects.

Neoplasm: An abnormal cellular proliferation, which includes benign andmalignant tumors, as well as other proliferative disorders.

Nucleic Acid: A deoxyribonucleotide or ribonucleotide polymer in eithersingle- or double-stranded form, and unless otherwise limited,encompasses known analogs of natural nucleotides that hybridize tonucleic acids in a manner similar to naturally occurring nucleotides.

Oligonucleotide or “Oligo”: Multiple nucleotides (i.e. moleculescomprising a sugar (e.g. ribose or deoxyribose) linked to a phosphategroup and to an exchangeable organic base, which is either a substitutedpyrimidine (Py) (e.g. cytosine (C), thymine (T) or uracil (U)) or asubstituted purine (Pu) (e.g. adenine (A) or guanine (G)). The term“oligonucleotide” as used herein refers to both oligoribonucleotides(ORNs) and oligodeoxyribonucleotides (ODNs). The term “oligonucleotide”also includes oligonucleosides (i.e. an oligonucleotide minus thephosphate) and any other organic base polymer. Oligonucleotides can beobtained from existing nucleic acid sources (e.g. genomic or cDNA), butare preferably synthetic (e.g. produced by oligonucleotide synthesis).

A “stabilized oligonucleotide” is an oligonucleotide that is relativelyresistant to in vivo degradation (for example via an exo- orendo-nuclease). In one embodiment, a stabilized oligonucleotide has amodified phosphate backbone. One specific, non-limiting example of astabilized oligonucleotide has a phosphothioate modified phosphatebackbone (wherein at least one of the phosphate oxygens is replaced bysulfur). Other stabilized oligonucleotides include: nonionic DNAanalogs, such as alkyl- and aryl-phosphonates (in which the chargedphosphonate oxygen is replaced by an alkyl or aryl group),phosphodiester and alkylphosphotriesters, in which the charged oxygenmoiety is alkylated. Oligonucleotides which contain a diol, such astetraethyleneglycol or hexaethyleneglycol, at either or both terminihave also been shown to be substantially resistant to nucleasedegradation.

An “immunostimulatory oligonucleotide,” “immunostimulatory CpGcontaining oligodeoxynucleotide,” “CpG ODN,” refers to anoligodeoxynucleotide, which contains a cytosine, guanine dinucleotidesequence and stimulates (e.g. has a mitogenic effect or induces cytokineproduction) vertebrate immune cells. The cytosine, guanine isunmethylated.

An “oligonucleotide delivery complex” is an oligonucleotide associatedwith (for example, ionically or covalently bound to; or encapsulatedwithin) a targeting means (such as a molecule that results in a higheraffinity binding to a target cell (B cell or natural killer (NK) cell)surface and/or increased cellular uptake by target cells). Examples ofoligonucleotide delivery complexes include oligonucleotides associatedwith: a sterol (for example, cholesterol), a lipid (for example,cationic lipid, virosome or liposome), or a target cell specific bindingagent (for example, a ligand recognized by a target cell specificreceptor). Preferred complexes must be sufficiently stable in vivo toprevent significant uncoupling prior to internalization by the targetcell. However, the complex should be cleavable or otherwise accessibleunder appropriate conditions within the cell so that the oligonucleotideis functional (Gursel, J. Immunol. 167:3324, 2001).

Pharmaceutical Agent or Drug: A chemical compound or composition capableof inducing a desired therapeutic or prophylactic effect when properlyadministered to a subject. Pharmaceutical agents include, but are notlimited to, chemotherapeutic agents and anti-infective agents.

Pharmaceutically Acceptable Carriers: The pharmaceutically acceptablecarriers useful in the methods and compositions disclosed herein areconventional. Remington's Pharmaceutical Sciences, by E. W. Martin, MackPublishing Co., Easton, Pa., 15th Edition (1975), describes compositionsand formulations suitable for pharmaceutical delivery of the fusionproteins herein disclosed.

In general, the nature of the carrier will depend on the particular modeof administration being employed. For instance, parenteral formulationsusually comprise injectable fluids that include pharmaceutically andphysiologically acceptable fluids such as water, physiological saline,balanced salt solutions, aqueous dextrose, glycerol or the like as avehicle. For solid compositions (e.g., powder, pill, tablet, or capsuleforms), conventional non-toxic solid carriers can include, for example,pharmaceutical grades of mannitol, lactose, starch, or magnesiumstearate. In addition to biologically-neutral carriers, pharmaceuticalcompositions to be administered can contain minor amounts of non-toxicauxiliary substances, such as wetting or emulsifying agents,preservatives, and pH buffering agents and the like, for example sodiumacetate or sorbitan monolaurate.

Salts encompassed within the term “pharmaceutically acceptable salts”refer to non-toxic salts. In one example, preparation of a salt, such asof an agonists of ADAM-10, are prepared by reacting the free base with asuitable organic or inorganic acid or by reacting the acid with asuitable organic or inorganic base.

Representative salts include the following salts: Acetate,Benzenesulfonate, Benzoate, Bicarbonate, Bisulfate, Bitartrate, Borate,Bromide, Calcium Edetate, Camsylate, Carbonate, Chloride, Clavulanate,Citrate, Dihydrochloride, Edetate, Edisylate, Estolate, Esylate,Fumarate, Gluceptate, Gluconate, Glutamate, Glycollylarsanilate,Hexylresorcinate, Hydrabamine, Hydrobromide, Hydrocloride,Hydroxynaphthoate, Iodide, Isethionate, Lactate, Lactobionate, Laurate,Malate, Maleate, Mandelate, Mesylate, Methylbromide, Methylnitrate,Methylsulfate, Monopotassium Maleate, Mucate, Napsylate, Nitrate,N-methylglucamine, Oxalate, Pamoate (Embonate), Palmitate, Pantothenate,Phosphate/diphosphate, Polygalacturonate, Potassium, Salicylate, Sodium,Stearate, Subacetate, Succinate, Tannate, Tartrate, Teoclate, Tosylate,Triethiodide, Trimethylammonium and Valerate.

Preventing or Treating a Disease: “Preventing” a disease refers toinhibiting the full development of a disease, for example in a personwho is known to have a predisposition to a disease, such as tumor or adisease caused by a pathogen, such as a virus or a bacteria. An exampleof a person with a known predisposition is someone with a history ofdiabetes in the family, or who has been exposed to factors thatpredispose the subject to a condition. “Treatment” refers to atherapeutic intervention that ameliorates a sign or symptom of a diseaseor pathological condition after it has begun to develop.

Purified: The term purified does not require absolute purity; rather, itis intended as a relative term. Thus, for example, a purified peptidepreparation is one in which the peptide or protein is more enriched thanthe peptide or protein is in its natural environment within a cell.Preferably, a preparation is purified such that the protein or peptiderepresents at least 50% of the total peptide or protein content of thepreparation.

Self-Complementary Nucleic Acid Sequence: A nucleic acid sequence thatcan form Watson-Crick base pairs. The four bases characteristic ofdeoxyribonucleic unit of DNA are the purines (adenine and guanine) andthe pyrimidines (cytosine and thymine). Adenine pairs with thymine viatwo hydrogen bonds, while guanine pairs with cytosine via three hydrogenbonds. If a nucleic acid sequence includes two or more bases in sequencethat can form hydrogen bonds with two or more other bases in the samenucleic acid sequence, then the nucleic acid includes aself-complementary sequence. In several embodiments, aself-complementary nucleic acid sequence includes 3, 4, 5, 6 or morebases that could form hydrogen bonds with 3, 4, 5, 6 or more bases,respectively, of the same nucleic acid sequence.

Therapeutically Effective Dose: A dose sufficient to preventadvancement, or to cause regression of the disease, or which is capableof relieving symptoms caused by the disease, such as pain or swelling.

Vaccine: A preparation of attenuated microorganisms (including but notlimited to bacteria and viruses), living microorganisms, antigen, orkilled microorganisms, administered for the prevention, amelioration ortreatment of infectious disease.

Unless otherwise explained, all technical and scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which this disclosure belongs. The singular terms“a,” “an,” and “the” include plural referents unless context clearlyindicates otherwise. Similarly, the word “or” is intended to include“and” unless the context clearly indicates otherwise. It is further tobe understood that all base sizes or amino acid sizes, and all molecularweight or molecular mass values, given for nucleic acids or polypeptidesare approximate, and are provided for description. Although methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of this disclosure, suitable methods andmaterials are described below. The term “comprises” means “includes.”All publications, patent applications, patents, and other referencesmentioned in this Detailed Description are incorporated by reference intheir entirety. In case of conflict, the present specification,including explanations of terms, will control. In addition, thematerials, methods, and examples are illustrative only and not intendedto be limiting.

II. Specific Embodiments

It is disclosed herein that agents that affect the activity and/orexpression of CXCL16 can be used in conjunction with a D-typeoligodeoxynucleotide (ODN). Specific compositions including one or moreD-type ODNs and an agent that modulates that activity and/or expressionof CXCL16 are provided herein. These compositions are of use to inducean immune response. The immune response can be to any antigen ofinterest, including by not limited to, an antigen from a pathogen or atumor.

A. D Oligodeoxynucletotides (ODNs)

D ODNs (also known as “A” class ODNs) differ both in structure andactivity from K ODNs (also known as “B” class ODNs) and a third type ofODNs, known as “C” class ODNs. For example, as disclosed herein, D ODNsstimulate the release of cytokines from cells of the immune system, andinduce the maturation of dendritic cells. In specific, non-limitingexamples D ODNs stimulate the release or production of IP-10 and IFN-αby monocytes and/or plasmacytoid dendritic cells.

With regard to structure, in one embodiment, a CpG motif in a D ODN hasbeen described by the formula:

5′ RY-CpG-RY 3′wherein the central CpG motif is unmethylated, R is A or G (a purine),and Y is C or T (a pyrimidine). D-type oligonucleotides include anunmethylated CpG dinucleotide. Inversion, replacement or methylation ofthe CpG reduces or abrogates the activity of the D oligonucleotide.

In one embodiment, a D-type ODN is at least about 16 nucleotides inlength and includes a sequence represented by the formula:

(SEQ ID NO: 21) 5′ X₁X₂X₃ Pu₁ Py₂ CpG Pu₃ Py₄ X₄X₅X₆(W)_(M) (G)_(N)-3′wherein the central CpG motif is unmethylated, Pu is a purinenucleotide, Py is a pyrimidine nucleotide, X and W are any nucleotide, Mis any integer from 0 to 10, and N is any integer from 4 to 10.

The region Pu₁ Py₂ CpG Pu₃ Py₄ is termed the CpG motif. The regionX₁X₂X₃ is termed the 5′ flanking region, and the region X₄X₅X₆ is termedthe 3′ flanking region. If nucleotides are included 5′ of X₁X₂X₃ in theD ODN, these nucleotides are termed the 5′ far-flanking region.Nucleotides 3′ of X₄X₅X₆ in the D ODN are termed the 3′ far-flankingregion.

In one specific, non-limiting example, Py₂ is a cytosine. In anotherspecific, non-limiting example, Pu₃ is a guanidine. In yet anotherspecific, non limiting example, Py₂ is a thymidine and Pu₃ is anadenine. In a further specific, non-limiting example, Pu₁ is an adenineand Py₂ is a tyrosine. In another specific, non-limiting example, Pu₃ isan adenine and Py₄ is a tyrosine.

In one specific, not limiting example, N is from about 4 to about 8. Inanother specific, non-limiting example, N is about 6.

In several embodiments, the D ODN is at least about 16 nucleotides inlength. For example, the D ODNs can be from about 16 to about 50nucleotides in length, or from about 18 to about 50 nucleotides inlength, or from about 18 to about 40 nucleotides in length, or fromabout 18 to about 30 nucleotides in length. Exemplary D ODNs aredisclosed below.

D ODNs can include modified nucleotides. Without being bound by theory,modified nucleotides can be included to increase the stability of a DODN. Without being bound by theory, because phosphothioate-modifiednucleotides confer resistance to exonuclease digestion, the D ODNs are“stabilized” by incorporating phosphothioate-modified nucleotides. Inone embodiment, the CpG dinucleotide motif and its immediate flankingregions include phosphodiester rather than phosphothioate nucleotides.In one specific, non-limiting example, the sequence Pu₁ Py₂ CpG Pu₃ Py₄includes phosphodiester bases. In another specific, non-limitingexample, all of the bases in the sequence Pu₁ Py₂ CpG Pu₃ Py₄ arephosphodiester bases. In yet another specific, non-limiting example,X₁X₂X₃ and X₄X_(S)X₆(W)_(M) (G)_(N) include phosphodiester bases. In yetanother specific, non-limiting example, X₁X₂X₃ Pu₁ Py₂ CpG Pu₃Py₄X₄X_(S)X₆(W)_(M) (G)_(N) (SEQ ID NO:21)include phosphodiester bases.In further non-limiting examples the sequence X₁X₂X₃ includes at mostone or at most two phosphothioate bases and/or the sequence X₄X₅X₆includes at most one or at most two phosphothioate bases. In additionalnon-limiting examples, X₄X₅X₆(W)_(M) (G)_(N) includes at least 1, atleast 2, at least 3, at least 4, or at least 5 phosphothioate bases.Thus, a D ODN can be a phosphothioate/phosphodiester chimera.

As disclosed herein, any suitable modification can be used to render theD ODN resistant to degradation in vivo (for example, via an exo- orendo-nuclease). In one specific, non-limiting example, a modificationthat renders the oligodeoxynucleotide less susceptible to degradation isthe inclusion of nontraditional bases such as inosine and quesine, aswell as acetyl-, thio- and similarly modified forms of adenine,cytidine, guanine, thymine, and uridine. Other modified nucleotidesinclude nonionic DNA analogs, such as alkyl or aryl phosphonates (i.e.,the charged phosphonate oxygen is replaced with an alkyl or aryl group,as set forth in U.S. Pat. No. 4,469,863), phosphodiesters andalkylphosphotriesters (i.e., the charged oxygen moiety is alkylated, asset forth in U.S. Pat. No. 5,023,243 and European Patent No. 0 092 574).Oligonucleotides containing a diol, such as tetraethyleneglycol orhexaethyleneglycol, at either or both termini, have also been shown tobe more resistant to degradation. The D-type oligodeoxynucleotides canalso be modified to contain a secondary structure (e.g., stem-loopstructure). Without being bound by theory, it is believed thatincorporation of a stem-loop structure renders an oligodeoxynucleotidemore effective.

In a further embodiment, Pu₁, Py₂ and Pu₃ Py₄ are self-complementary. Inanother embodiment, X₁X₂X₃ and X₄X₅X₆ are self-complementary. In yetanother embodiment X₁X₂X₃Pu₁ Py₂ and Pu₃ Py₄ X₄X₅X₆ areself-complementary.

Specific non-limiting examples of a D ODN wherein Pu₁ Py₂ and Pu₃ Py₄are self-complementary include, but are not limited to, ATCGAT, ACCGGT,ATCGAC, ACCGAT, GTCGAC, or GCCGGC (wherein the CpG is underlined).Without being bound by theory, the self-complementary base sequences canhelp to form a stem-loop structure with the CpG dinucleotide at the apexto facilitate immunostimulatory functions. Thus, in one specific,non-limiting example, D ODNs wherein Pu₁ Py₂ and Pu₃ Py₄ areself-complementary induce higher levels of IFN-γ production from a cellof the immune system. The self-complementary need not be limited to Pu₁Py₂ and Pu₃ Py₄. Thus, in another embodiment, additional bases on eachside of the three bases on each side of the CpG-containing hexamer forma self-complementary sequence (see above).

One specific, non-limiting example of a sequence wherein Pu₁ Py₂ and Pu₃Py₄ are self-complementary but wherein the far-flanking sequences arenot self-complementary is

GGTGCATCGATACAGGGGGG (DV113, SEQ ID NO: 6, see Table 1)

This oligodeoxynucleotide has a far-flanking region that is notself-complementary and induces high levels of IFN-γ and IFN-α.

Another specific, non-limiting example of a D ODN is:

GGTGCGTCGATGCAGGGGGG (DV28, SEQ ID NO: 3, see Table 1)

This D ODN is of use for inducing production and/or release of cytokinesfrom immune cells, although it lacks a self-complementary motif.

In one embodiment, the D ODNs are at least about 16 nucleotides inlength. In a second embodiment, a D ODN is at least about 18 nucleotidesin length. In another embodiment, a D ODN is from about 16 nucleotidesin length to about 100 nucleotides in length. In yet another embodiment,a D ODN is from about 16 nucleotides in length to about 50 nucleotidesin length. In a further embodiment, a D ODN is from about 18 nucleotidesin length to about 30 nucleotides in length.

In another embodiment, the D ODN is at least 18 nucleotides in length,and at least two G's are included at the 5′ end of the molecule, suchthat the oligodeoxynucleotide includes a sequence represented by theformula:

5′ GGX₁X₂X₃ Pu₁ Py₂ CpG Pu₃ Py₄ X₄X₅X₆(W)_(M) (G)_(N)-3′.

The D ODN can include additional G's at the 5′ end of theoligodeoxynucleotide. In one specific example, about 1 or about 2 G'sare included at the 5′ end of an oligodeoxynucleotide including asequence as set forth as the above formula.

Examples of a D ODN include, but are not limited to the sequence shownin the following table:

TABLE 1* ODN D ODN SEQUENCE SEQUENCE IDENTIFIER DV104GGTGCATCGATGCAGGGGGG (SEQ ID NO: 1) DV19 GGTGCATCGATGCAGGGGGG(SEQ ID NO: 1) DV29 GGTGCACCGGTGCAGGGGGG (SEQ ID NO: 2) DV35GGTGCATCGATGCAGGGGGG (SEQ ID NO: 1) DV28 GGTGCGTCGATGCAGGGGGG(SEQ ID NO: 3) DV106 GGTGTGTCGATGCAGGGGGG (SEQ ID NO: 4) DV116TGCATCGATGCAGGGGGG (SEQ ID NO: 5) DV113 GGTGCATCGATACAGGGGGG(SEQ ID NO: 6) DV34 GGTGCATCGATGCAGGGGGG (SEQ ID NO: 7) DV102GGTGCATCGTTGCAGGGGGG (SEQ ID NO: 8) DV32 GGTGCGTCGA CGCAGGGGGG(SEQ ID NO: 9) DV117 GGTCGATCGATGCACGGGGG (SEQ ID NO: 10) DV37 GGTGCATCGAT GCAGGGGGG (SEQ ID NO: 11) DV25 GGTGCATCGATGCAGGGGGG(SEQ ID NO: 11) DV30 GGTGCATCGACGCAGGGGGG (SEQ ID NO: 12) dv120GGTGCATCGATAGGCGGGGG (SEQ ID NO: 13) DV27 GGTGCACCGATGCAGGGGGG(SEQ ID NO: 14) dvl19 CCTGCATCGATGCAGGGGGG (SEQ ID NO: 15) D142GGTATATCGATATAGGGGGG (SEQ ID NO: 16) d143 GGTGGAT CG ATCCAGGGGGG(SEQ ID NO: 17) Underlined bases are phosphodiester. *indicatesmethylated CG. Bold indicates self-complementary sequences. Sequenceidentifier is noted below the nucleic acid sequence.

Additional exemplary D ODN sequences can be found in U.S. patentapplication Ser. No. 10/068,160, and in Verthelyi et al., J. Immunol.166:2372-2377, 2001, which are both herein incorporated by reference intheir entireties. D ODN can be used in combination to induce an immuneresponse. Thus, multiple D ODNs can be utilized to induce an immuneresponse. For example, two, three, four, five or more D ODNs can beutilized to induce an immune response. In addition, a single ODN can begenerated that includes the two or more D ODN CpG motifs disclosedherein.

The D ODN can be synthesized de novo using any of a number of procedureswell known in the art. For example, the oligodeoxynucleotides can besynthesized as set forth in U.S. Pat. No. 6,194,388, which is hereinincorporated by reference in its entirety. A D ODN can be synthesizedusing, for example, the B-cyanoethyl phosphoramidite method ornucleoside H-phosphonate method. These chemistries can be performed by avariety of automated oligonucleotide synthesizers available in themarket. Alternatively, oligodeoxynucleotides can be prepared fromexisting nucleic acid sequences (e.g. genomic or cDNA) using knowntechniques, such as employing restriction enzymes, exonucleases orendonucleases, although this method is less efficient than directsynthesis.

The response elicited by D, K and C ODNs is dependent on Tol-likereceptor 9 (TLR9). Cells lacking TLR9 are unresponsive to any form ofCpG ODNs. It is disclosed herein that an additional receptor is requiredfor optimal recognition of D ODN, namely CXCL16. Altering the expressionof activity of this receptor can be used to alter an immune responseinduced by a D ODN.

Methods of Altering an Immune Response

A method is disclosed herein for altering the uptake of a D ODN.Increasing the uptake of a D ODN can be used to increase theimmunostimulatory activity of the D ODN. Similarly, decreasing theuptake of a D ODN can be used to attenuate the immunostimulatoryactivity of the D ODN.

As described above, D ODNs are of use in producing an immune response(see also PCT Publication Nos. WO 0061151A3, WO9956755A1, WO9840100A1,WO9818810A1, WO0122990A2, which are all herein incorporated by referencein their entirety). Administration of a D ODN can be by any suitablemethod, including in vivo or ex vivo administration. For example, a DODN can be used to stimulate monocytes and/or natural killer cells,and/or to induce the maturation of dendritic cells. Furthermore, a D ODNcan be used to increase the production of cytokines (for example IP-10,IFN-α or IFN-γ) by a cell of the immune system. D ODNs can be used toinduce a T cell response to an antigen of interest. D ODNs are also ofuse in producing an immune response against pathogens (such asbacterial, viral, or fungal pathogens). D ODNs can be used to induce aprotective immune response. D ODNs can be used to increase an immuneresponse to a tumor antigen. Thus, D ODNs are of use in a variety oftherapeutic applications, and can also be utilized in vaccineformulations.

An agent that increases the activity and/or expression of CXCL16 can beused to increase one or more immune response induced by a D ODN. In oneembodiment, a method is provided for increasing the uptake of a D ODN.The method includes administering an effective amount of an agent thatincreases the activity and/or expression of CXCL16. In anotherembodiment, a method is disclosed herein for inducing an immune responsein a subject by administering a D ODN and an agent that increases theactivity and/or expression of CXCL16. The immune response can include,but is not limited to, induction of the maturation of a dendritic cellor the activation of a natural killer cell and/or a monocyte. The immuneresponse can also include the production of a cytokine, such as, forexample, IL-10, IP-10, IFN-α or IFN-γ. The immune response can alsoinclude an immune response against an antigen, such as a bacterial,viral, or fungal antigen. The immune response can include an immuneresponse to a tumor antigen.

In one example, a D ODN is administered in conjunction with an agentthat increases the expression and/or activity of CXCL16 to a subjectthat has an autoimmune disease. Exemplary autoimmune diseases affectingmammals include rheumatoid arthritis, juvenile oligoarthritis,collagen-induced arthritis, adjuvant-induced arthritis, Sjogren'ssyndrome, multiple sclerosis, experimental autoimmune encephalomyelitis,inflammatory bowel disease (e.g., Crohn's disease, ulcerative colitis),autoimmune gastric atrophy, pemphigus vulgaris, psoriasis, vitiligo,type 1 diabetes, non-obese diabetes, myasthenia gravis, Grave's disease,Hashimoto's thyroiditis, sclerosing cholangitis, sclerosingsialadenitis, systemic lupus erythematosis, autoimmune thrombocytopeniapurpura, Goodpasture's syndrome, Addison's disease, systemic sclerosis,polymyositis, dermatomyositis, autoimmune hemolytic anemia, perniciousanemia, and the like. Specific, non-limiting examples of autoimmunediseases include, but are not limited to diabetes, rheumatoid arthritis,lupus erythematosus, and multiple sclerosis.

In another example, a D ODN is administered in conjunction with an agentthat increases the expression and/or activity of CXCL16 to treat,prevent, or ameliorate an allergic reaction in a subject. An allergyrefers to an acquired hypersensitivity to a substance (an allergen).Allergic conditions include eczema, allergic rhinitis or coryza, hayfever, bronchial asthma, uticaria (hives), food allergies, and otheratopic conditions. The list of allergens is extensive and includespollens, insect venoms, animal dander, dust, fungal spores, and drugs(such as antibiotics like penicillin or tetracycline). Examples ofnatural, animal, and plant allergens can be found in PCT Publication No.WO 98/18810. In one embodiment a D ODN is administered to a subject inconjunction with an agent that increases the expression and/or activityof CXCL16 to treat an allergic condition such as allergic asthma. The DODN and the agent that increases the uptake and/or activity of CXCL16can also be administered in combination with an anti-allergenic agent.Suitable anti-allergenic agents include those substances given intreatment of the various allergic conditions described above, examplesof which can be found in the Physicians' Desk Reference (1998).

In a further example, a D ODN is administered to a subject that has aneoplasm. In one embodiment, the subject has cancer. The D ODN isadministered in conjunction with an agent that increases the expressionand/or activity of CXCL16. The D ODN and the agent that increases theexpression and/or activity of CXCL16 can be administered either alone orin combination with any suitable anti-neoplastic agent, such as achemotherapeutic agent or radiation. Suitable neoplasms include benignand malignant cancer. The neoplasm can be from any origin, and include,but are not limited to, solid tumors such as cancers of the brain, lung(e.g., small cell and non-small cell), ovary, breast, prostate, liver,lung, skin, and colon, as well as carcinomas and sarcomas. The neoplasmcan also be a lymphoma or a leukemia. Examples of hematological tumorsinclude leukemias, including acute leukemias (such as acute lymphocyticleukemia, acute myelocytic leukemia, acute myelogenous leukemia andmyeloblastic, promyelocytic, myelomonocytic, monocytic anderythroleukemia), chronic leukemias (such as chronic myelocytic(granulocytic) leukemia, chronic myelogenous leukemia, and chroniclymphocytic leukemia), polycythemia vera, lymphoma, Hodgkin's disease,non-Hodgkin's lymphoma (indolent and high grade forms), multiplemyeloma, Waldenstrom's macroglobulinemia, heavy chain disease,myelodysplastic syndrome, and myelodysplasia.

Examples of solid tumors, such as sarcomas and carcinomas, includefibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenicsarcoma, and other sarcomas, synovioma, mesothelioma, Ewing's tumor,leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, lymphoid malignancy,pancreatic cancer, breast cancer, lung cancers, ovarian cancer, prostatecancer, hepatocellular carcinoma, squamous cell carcinoma, basal cellcarcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous glandcarcinoma, papillary carcinoma, papillary adenocarcinomas, medullarycarcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bileduct carcinoma, choriocarcinoma, Wilms' tumor, cervical cancer,testicular tumor, bladder carcinoma, and CNS tumors (such as a glioma,astrocytoma, medulloblastoma, craniopharyogioma, ependymoma, pinealoma,hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma,melanoma, neuroblastoma and retinoblastoma). The administration of the DODN and the agent that increases the expression and/or activity ofCXCL16 can be used to reduce tumor burden.

In yet another example, a method is provided to enhance the efficacy ofany suitable vaccine. The method includes the administration of a D ODNin conjunction with an agent that increases the expression and/oractivity of CXCL16 and a vaccine component. Suitable vaccines includethose directed against Leishmania, Hepatitis A, B, and C, examples ofwhich can be found in the Physicians' Desk Reference (1998), and DNAvaccines directed against, for example, malaria. (See generally Klinmanet al., Vaccine 17:19, 1999; McCluskie and Davis, J. Immun. 161:4463,1998). The vaccine can be a subunit vaccine, or can include anattenuated or heat-killed virus.

In an additional example, D ODN and the agent that induces theexpression and/or activity CXCL16 can be used to treat or ameliorate anycondition associated with an infectious agent. Thus, the D ODN and theagent that increases the activity and/or expression of CXCL16 can beadministered to a subject infected with the infectious agent. Specific,non-limiting examples of conditions associated with infectious agentsare tularemia, francisella, schistosomiasis, tuberculosis, malaria, andleishmaniasis. Examples of infectious agents are viruses, bacteria,fungi, and other organisms (such as protists) can be found in PCTPublication No. WO 98/18810. The D ODN and the agent that induces theexpression and/or activity of CXCL16 can be administered in combinationwith any suitable anti-infectious agent, such as an antiviral,anti-fungal or anti-bacterial agent (see Physicians' Desk Reference,1998).

The agent that increases the activity and/or expression of CXCL16 can beone or more cytokines. The cytokines interferon gamma (IFN-γ, see, forexample GENBANK® Accession No. AAM28885, May 16, 2002) and tumornecrosis factor alpha (TNF-α, see, for example GENBANK® Accession No.NP_(—)000585, Aug. 20, 2006, incorporated herein by reference) induceCXCL16, either alone or in combination. Thus, IFN-γ and/or TNF-α couldbe used to increase the uptake of a D ODN. Nucleic acids encoding IFN-γor TNF-α could be administered to the subject in order increase theuptake of a D ODN. The agents that induce the expression and/or activityof CXCL16 include nucleic acids encoding CXCL16, such as human CXCL16.

Agents that increase the activity and/or expression of CXCL16 include,but are not limited to, antagonists of ADAM-10. Exemplary antagonists ofADAM-10 are GW280264X, G1254023X or GM6001. GM6001 has the chemicalformula C₂₀H₂₈N₄O₄, and has the structure shown below:

GW280264X ((2R,3S)-3-(formyl-hydroxyamino)-2-(2-methyl-1-propyl)hexanoic acid[(1S)-5-benzyloxycarbamoylamino-1-(1,3-thiazol-2-ylcarbamoyl)-1-pentyl]amide)and G1254023X((2R,3S)-3-(formyl-hydroxyamino)-2-(3-phenyl-1-propyl)butanoicacid[(1S)-2,2-dimethyl-1-methylcarbamoyl-1-propyl]amide) can besynthesized as described in U.S. Pat. Nos. 6,172,064, 6,191,150 and6,329,400. The compounds can be administered as a single or polymorphiccrystalline form or forms, an amorphous form, a single enantiomer, aracemic mixture, a single stereoisomer, a mixture of stereoisomers, asingle diastereoisomer, a mixture of diastereoisomers, a solvate, apharmaceutically acceptable salt, a solvate, a prodrug, abiohydrolyzable ester, or a biohydrolyzable amide thereof. Thesecompounds can also be administered as pharmaceutically acceptable salt,solvate, biohydrolyzable ester, biohydrolyzable amide, affinity reagent,or prodrug thereof for use in therapy.

Other MMP-2 inhibitors are of use, such as batimastat. In addition, aninhibitor such as inhibitor of the formula C₁₈H₃₅NO₂:

-   Also of use is an inhibitor of the formula C₁₃H₁₄N₄O₃S₂:

-   Also of use is an inhibitor of the formula C₂₁H₂₃N₇O₂S₂:

-   Also of use is an inhibitor of the formula C₂₁H₁₉NO₄S:

Additional agents can be administered in conjunction with the D ODN andthe agent that alters the activity and/or expression of CXCL16. Theseagents include a protein, an antigenic epitope, a hydrocarbon, lipid,mitogen, an anti-infectious agent (such as antiviral, antifungal, oranti-bacterial agent), a chemotherapeutic agent or a vaccine (such as alive, attenuated, or heat-killed vaccine). Additional agents can beadministered simultaneously or sequentially with the D ODN and the agentthat increases the activity and/or expression of CXCL16.

In one embodiment, a method is provided for activating an antigenpresenting cell or lymphocyte in vitro. The method includes contacting amonocyte or a dendritic cell precursor in vitro with a D ODN and anagent that increases CXCL16 expression and/or activity to produce anactivated antigen presenting cell. The monocytes or dendritic cellprecursors can be contacted with the D ODN and the agent that increasesCXCL16 expression and/or activity in the presence of or in the absenceof an antigen. The activated antigen presenting cell can be administeredto the subject to induce an immune response. Alternatively, lymphocytesor natural killer cells are contacted with the activated antigenpresenting cells in vitro, or with cytokines secreted by the activatedantigen presenting cells in vitro, to produce activated lymphocytes oractivated natural killer cells. The activated lymphocytes or naturalkiller cells can then be administered to the subject to induce an immuneresponse.

In another embodiment, a method is provided for decreasing the uptake ofa D ODN. The method includes providing an agent that decreases theactivity and/or expression of CXCL16. In one example, the agent thatdecreases the uptake of a D ODN is ionomycin. In another example, theagent that decreases the activity and/or expression of CXCL16 is anantisense oligonucleotide, small inhibitory mRNA (simRNA) or a ribozymethat cleaves CXCL16 mRNA. One of skill in the art can readily producethese molecules using the CXCL nucleic acid sequence and/or proteinsequence.

In a further example, the agent that decreases the activity and/orexpression of CXCL16 is an antibody. The antibody or antibody fragmentcan be a humanized immunoglobulin. Generally, the humanizedimmunoglobulin specifically binds to the CXCL16, or a molecule thatregulates CXCL16, with an affinity constant of at least 10⁷ M⁻¹, such asat least 10⁸ M⁻¹ or 10⁹ M⁻¹. The use of antibody components derived fromhumanized monoclonal antibodies obviates potential problems associatedwith the immunogenicity of the constant regions of the donor antibody.Techniques for producing humanized monoclonal antibodies are described,for example, by Jones, et al., Nature 321:522, 1986; Riechmann, et al.,Nature 332:323, 1988; Verhoeyen, et al., Science 239:1534, 1988; Carter,et al., Proc. Nat'l Acad. Sci. U.S.A. 89:4285, 1992; Sandhu, Crit. Rev.Biotech. 12:437, 1992; and Singer, et al., J. Immunol. 150:2844, 1993.

Antibodies include intact molecules as well as fragments thereof, suchas Fab, F(ab′)₂, and Fv which include a heavy chain and light chainvariable region and are capable of binding the epitopic determinant.These antibody fragments retain some ability to selectively bind withtheir antigen or receptor and are defined as follows: (1) Fab, thefragment which contains a monovalent antigen-binding fragment of anantibody molecule, can be produced by digestion of whole antibody withthe enzyme papain to yield an intact light chain and a portion of oneheavy chain; (2) Fab′, the fragment of an antibody molecule can beobtained by treating whole antibody with pepsin, followed by reduction,to yield an intact light chain and a portion of the heavy chain; twoFab′ fragments are obtained per antibody molecule; (3) (Fab′)₂, thefragment of the antibody that can be obtained by treating whole antibodywith the enzyme pepsin without subsequent reduction; F(ab′)₂ is a dimerof two Fab′ fragments held together by two disulfide bonds; (4) Fv, agenetically engineered fragment containing the variable region of thelight chain and the variable region of the heavy chain expressed as twochains; and (5) Single chain antibody (such as scFv), defined as agenetically engineered molecule containing the variable region of thelight chain, the variable region of the heavy chain, linked by asuitable polypeptide linker as a genetically fused single chainmolecule.

Methods of making these fragments are known in the art (see for example,Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring HarborLaboratory, New York, 1988). An epitope is any antigenic determinant onan antigen to which the paratope of an antibody binds. Epitopicdeterminants usually consist of chemically active surface groupings ofmolecules such as amino acids or sugar side chains and usually havespecific three dimensional structural characteristics, as well asspecific charge characteristics

Agents that increase intracellular calcium are of use in decreasingCXCL16 activity. Without being bound by theory, agents that increaseintracellular calcium are of use in increasing ADAM-10 activityresulting in increased cleavage of CXCL-16. For example, a cross-linkingagent can be used to cross link the BDCA-2 receptor on the surface ofplasmacytoid dendritic cells. This results in increased intracellularcalcium flux and a corresponding decrease in CXCL16 activity.Plasmacytoid dendritic cells treated with a cross-linking agent fail toproduce interferon alpha in response to a D ODN.

CXCL16 plays a role in atherosclerosis and Alzheimer's disease. Thus,reduced levels of CXCL16 can be used to treat subjects withatherosclerosis and Alzheimer's disease. Reducing CXCL16 on the surfaceof plasmacytoid dendritic cells can also be effective in treating SLE.Thus, a method is provided herein for the treatment of subjects withatherosclerosis, Alzheimer's disease, and systemic lupus Erythematosus.In several examples, the method includes administering an antibody thatspecifically binds CXCL16, an agent that increases the activity ofADAM-10, such as ionomycin.

For use in vivo, generally a pharmaceutical composition including atherapeutically effective amount of the agent that alters CXCL16expression and/or activity is administered to the subject of interest. AD ODN is also administered to the subject of interest. In one example,the agent that alters CXCL16 expression and/or activity can be includedin the same composition as the D ODN. Thus, compositions are providedherein that include a therapeutically effective amount agent that altersCXCL16 and a therapeutically effective amount D ODN in apharmaceutically acceptable carrier. Optionally, additional therapeuticagents can be included, such as, but not limited to chemotherapeuticagents, antigens, attenuated or heat killed virus, and/or cytokines.However, the CXCL16 can be included in a first composition, and the DODN included in a second composition, and the two compositionsadministered to a subject of interest sequentially.

The pharmaceutically acceptable carriers and excipients useful in thisdisclosure are conventional. For instance, parenteral formulationsusually comprise injectable fluids that are pharmaceutically andphysiologically acceptable fluid vehicles such as water, physiologicalsaline, other balanced salt solutions, aqueous dextrose, glycerol or thelike. Excipients that can be included are, for instance, proteins, suchas human serum albumin or plasma preparations. If desired, thepharmaceutical composition to be administered may also contain minoramounts of non-toxic auxiliary substances, such as wetting oremulsifying agents, preservatives, and pH buffering agents and the like,for example sodium acetate or sorbitan monolaurate.

The dosage form of the pharmaceutical composition will be determined bythe mode of administration chosen. Agents that increase the expressionand/or activity of CXCL-16 and D ODN can be administered systemically orlocally. The agents can be administered in oral (including buccal andsublingual) dosage forms as tablets, capsules (each including timedrelease and sustained release formulations), pills, powders, granules,elixirs, tinctures, suspensions, syrups and emulsions. Likewise, theymay also be administered in nasal, ophthalmic, otic, rectal, topical,intravenous (both bolus and infusion), intraperitoneal, intraarticular,subcutaneous or intramuscular inhalation form, all using forms wellknown to those of ordinary skill in the art.

For instance, in addition to injectable fluids, topical and oralformulations can be employed. Topical preparations can include eyedrops, ointments, sprays and the like. Oral formulations may be liquid(e.g., syrups, solutions, or suspensions), or solid (e.g., powders,pills, tablets, or capsules). For solid compositions, conventionalnon-toxic solid carriers can include pharmaceutical grades of mannitol,lactose, starch, or magnesium stearate. Actual methods of preparing suchdosage forms are known, or will be apparent, to those of ordinary skillin the art.

For example, oral dosages of antagonists of ADAM-10, when used for theindicated effects, will range between about 0.1 to 2000 mg/kg of bodyweight per day, and particularly 1 to 1000 mg/kg of body weight per day.Oral dosage units are generally administered in the range of from 1 toabout 250 mg, such as from about 25 to 250 mg. The daily dosage for a 70kg mammal can be, for example, in the range of about 10 mg to 5 grams ofa compound. The dosage regimen is generally selected in accordance witha variety of factors including type, species, age, weight, sex andmedical condition of the patient; the severity of the condition to betreated; the route of administration; the renal and hepatic function ofthe patient; and the particular compound or salt thereof employed. Anordinarily skilled physician or veterinarian can readily determine andprescribe a therapeutically effective amount.

In some embodiments, pharmaceutical compositions will be formulated inunit dosage form, suitable for individual administration of precisedosages. The amount of active compound(s) administered will be dependenton the subject being treated, the severity of the affliction, and themanner of administration, and is best left to the judgment of theprescribing clinician. Within these bounds, the formulation to beadministered will contain a quantity of the active component(s) inamounts effective to achieve the desired effect in the subject beingtreated. See for example, U.S. Pat. No. 6,172,064, for a discussion ofthe preparation of tablets capsules, suppositories, formulations forinjection, and formulations for inhalation therapy, such as for use withADAM-10 inhibitors.

Compositions can be formulated with an appropriate solid or liquidcarrier, depending upon the particular mode of administration chosen. Ifdesired, the disclosed pharmaceutical compositions can also containminor amounts of non-toxic auxiliary substances, such as wetting oremulsifying agents, preservatives, and pH buffering agents and the like,for example sodium acetate or sorbitan monolaurate. Excipients that canbe included in the disclosed compositions include flow conditioners andlubricants, for example silicic acid, talc, stearic acid or saltsthereof, such as magnesium or calcium stearate, and/or polyethyleneglycol, or derivatives thereof.

The compositions can be provided as parenteral compositions, such as forinjection or infusion. Such compositions are formulated generally bymixing a disclosed therapeutic agent at the desired degree of purity, ina unit dosage injectable form (solution, suspension, or emulsion), witha pharmaceutically acceptable carrier, for example one that is non-toxicto recipients at the dosages and concentrations employed and iscompatible with other ingredients of the formulation. In addition, adisclosed therapeutic agent can be suspended in an aqueous carrier, forexample, in an isotonic buffer solution at a pH of about 3.0 to about8.0, preferably at a pH of about 3.5 to about 7.4, 3.5 to 6.0, or 3.5 toabout 5.0. Useful buffers include sodium citrate-citric acid and sodiumphosphate-phosphoric acid, and sodium acetate/acetic acid buffers. Theactive ingredient, optionally together with excipients, can also be inthe form of a lyophilisate and can be made into a solution prior toparenteral administration by the addition of suitable solvents.Solutions such as those that are used, for example, for parenteraladministration can also be used as infusion solutions.

Therapeutic compositions can be formulated in unit dosage form, suitablefor individual administration of precise dosages. In pulse doses, abolus administration is provided, followed by a time-period wherein nodisclosed agent that affects CXCL16 activity/ D ODN is administered tothe subject, followed by a second bolus administration. Atherapeutically effective amount of the composition can be administeredin a single dose, or in multiple doses, for example daily, during acourse of treatment. In specific, non-limiting examples, pulse doses areadministered during the course of a day, during the course of a week, orduring the course of a month.

A form of repository or “depot” slow release preparation can be used sothat therapeutically effective amounts of the preparation are deliveredinto the bloodstream over many hours or days following transdermalinjection or delivery. Such long acting formulations can be administeredby implantation (for example subcutaneously or intramuscularly) or byintramuscular injection. The compounds can be formulated with suitablepolymeric or hydrophobic materials (for example as an emulsion in anacceptable oil) or ion exchange resins, or as sparingly solublederivatives, for example, as a sparingly soluble salt.

The therapeutic compositions that can be delivered by way of a pump (seeLanger, supra; Sefton, CRC Crit. Ref. Biomed. Eng. 14:201, 1987;Buchwald et al., Surgery 88:507, 1980; Saudek et al., N. Engl. J. Med.321:574, 1989) or by continuous subcutaneous infusions, for example,using a mini-pump. An intravenous bag solution can also be employed. Onefactor in selecting an appropriate dose is the result obtained, asmeasured by the methods disclosed here, as are deemed appropriate by thepractitioner. Other controlled release systems are discussed in Langer(Science 249:1527-33, 1990).

In one example, a pump is implanted (for example see U.S. Pat. Nos.6,436,091; 5,939,380; and 5,993,414). Implantable drug infusion devicesare used to provide patients with a constant and long-term dosage orinfusion of a therapeutic agent. Such device can be categorized aseither active or passive.

Active drug or programmable infusion devices feature a pump or ametering system to deliver the agent into the patient's system. Anexample of such an active infusion device currently available is theMedtronic SYNCHROMED™ programmable pump. Passive infusion devices, incontrast, do not feature a pump, but rather rely upon a pressurized drugreservoir to deliver the agent of interest. An example of such a deviceincludes the Medtronic ISOMED™.

In particular examples, compositions including an agent that affectsCXCL16 activity and/or expression are administered by sustained-releasesystems. The D ODN can also be administered by sustained releasesystems, alone or in combination with the agent that affects CXCL16activity and/or expression. Suitable examples of sustained-releasesystems include suitable polymeric materials (such as, semi-permeablepolymer matrices in the form of shaped articles, for example films, ormirocapsules), suitable hydrophobic materials (for example as anemulsion in an acceptable oil) or ion exchange resins, and sparinglysoluble derivatives (such as, for example, a sparingly soluble salt).Sustained-release compositions can be administered orally, parenterally,intracistemally, intraperitoneally, topically (as by powders, ointments,gels, drops or transdermal patch), or as an oral or nasal spray.Sustained-release matrices include polylactides (U.S. Pat. No.3,773,919, EP 58,481), copolymers of L-glutamic acid andgamma-ethyl-L-glutamate (Sidman et al., Biopolymers 22:547-556, 1983,poly(2-hydroxyethyl methacrylate)); (Langer et al., J. Biomed. Mater.Res. 15:167-277, 1981; Langer, Chem. Tech. 12:98-105, 1982, ethylenevinyl acetate (Langer et al., Id.) or poly-D-(−)-3-hydroxybutyric acid(EP 133,988).

Polymers can be used for ion-controlled release. Various degradable andnondegradable polymeric matrices for use in controlled drug delivery areknown in the art (Langer, Accounts Chem. Res. 26:537, 1993). Forexample, the block copolymer, polaxamer 407 exists as a viscous yetmobile liquid at low temperatures but forms a semisolid gel at bodytemperature. It has shown to be an effective vehicle for formulation andsustained delivery of recombinant interleukin-2 and urease (Johnston etal., Pharm. Res. 9:425, 1992; and Pec, J. Parent. Sci. Tech. 44(2):58,1990). Alternatively, hydroxyapatite has been used as a microcarrier forcontrolled release of proteins (Ijntema et al., Int. J. Pharm. 112:215,1994). In yet another aspect, liposomes are used for controlled releaseas well as drug targeting of the lipid-capsulated drug (Betageri et al.,Liposome Drug Delivery Systems, Technomic Publishing Co., Inc.,Lancaster, Pa., 1993). Numerous additional systems for controlleddelivery of therapeutic proteins are known (for example, U.S. Pat. Nos.5,055,303; 5,188,837; 4,235,871; 4,501,728; 4,837,028; 4,957,735; and5,019,369; 5,055,303; 5,514,670; 5,413,797; 5,268,164; 5,004,697;4,902,505; 5,506,206; 5,271,961; 5,254,342; and 5,534,496).

Screening for Agents that Alter an Immune Response

An in vitro method is provided herein for screening for an agent thataffects the immune response induced by a D ODN. In one example, themethod includes contacting a cell with an agent in vitro, and assessingthe expression and/or activity of CXCL16. An increase in the expressionand/or activity of CXCL16 indicates that the agent is of use inincreasing the uptake of a D ODN. An increase in the expression oractivity of CXCL16 also indicates that the agent will increase an immuneresponse induced by an ODN. A decrease in the expression and/or activityof CXCL16 indicates that the agent is of use in decreasing the uptake ofa D ODN. A decrease in the expression or activity of CXCL16 alsoindicates that the agent will decrease an immune response induced by anODN.

A parameter of an immune response can also be measured. In oneembodiment, cytokine production is measured. For example, the productionof interferon-α or IL-6 can be measured. In another embodiment,expression of a protein is assess, such as, but not limited to, theexpression of HLA.

The expression or activity of CXCL16 can be measured by any method knownto those of skill in the art. For example, the expression of CXCL16 canbe measured by polymerase chain reaction, Western blot, and ELISA andflow cytometry (see Abel et al., J. Immunol. 172:6362-6372, 2004,incorporated herein by reference for a detailed description of thesemethods).

One exemplary non-limiting method for measuring CXCL16 is reversetranscriptase polymerase chain reaction (RT-PCR). One exemplarynon-liming method utilizes RNA isolated from cultured cells using TRIzolreagent. Total RNA (1 μg) is treated with DNaseI and is reversetranscribed to cDNA using a suitable commercially available polymerase.For each sample, a control without polymerase is run parallel to allowassessment of genomic DNA contamination. Each cDNA sample is analyzedfor expression of CXCL16 and GAPDH by real-time quantitative RT-PCRusing the fluorescent TAQMAN™ 5′-nuclease assay. Exemplary sequences offorward primer, reverse primer, and probe for CXCL16 are 5′-GAG CTC ACTCGT CCC AAT GAA-3′ (SEQ ID NO: 18), 5′-TCA GGC CCA ACT GCC AGA C-3′ (SEQID NO: 19), and 5′-FAM CAC CAT TCA CAC TGC GGG CCA C TAMRA-3′ (SEQ IDNO: 20). Levels of CXCL16 mRNA can be quantified by comparison of thefluorescence of each sample with that of a serially diluted standard ofhuman genomic DNA.

One exemplary and non-limiting method for an ELISA assay for a CXCL16 isas follows: Microlon 96-well plates (Greiner, Nurtingen, Germany) atroom temperature, are utilized with a reaction volume was 50 μl. Theplate is coated overnight with 2 mg/ml goat anti-human CXCL16 in 50 mMNa₂CO₃ (pH9.3), washed three times with 0.05% TWEEN in phosphatebuffered saline (PBS-T), and blocked with PBS-T containing 2% bovineserum album (BSA) for 2 hours. The plate is dried and the samples areadded for 2 hours. A standard prepared as nine serial ½ dilutions of1.25 nM recombinant human CXCL16 in either PBS-T with 1% BSA, serum-freemedium, or cell lysis buffer is run in parallel. Following washing, 200ng/ml biotinylated rabbit anti-human CXCL16 in PBS-T/1% BSA is added toeach well and the plate is incubated at room temperature for 1 hour.After washing, 100 mU/ml streptavidin-peroxidase conjugate (Roche) inPBS-T/1% BSA is added for 1 hour. After washing, chromogenic peroxidasesubstrate is added. The reaction is stopped after a twenty-minuteincubation by addition of 1.8 M H₂SO₄ before the optical density (OD)was determined at 450 nm.

One exemplary and non-limiting method for FACS analysis of CXCL16expression is as follows: cells are suspended in ice-cold PBS containing0.1% BSA and 0.01% NaN₃ at 3×10⁵ cells/ml and incubated with purifiedrabbit anti-human CXCL16 or rabbit IgG control (both at 2 μg/ml in PBSwith 0.1% BSA and 0.01% NaN₃) for 1 hour on ice. Following two-foldwashing, cells are incubated with secondary fluorescein-conjugated goatanti-rabbit IgG for 1 hour on ice. Cells are washed twice and suspendedin ice-cold PBS containing 2% paraformaldehyde. The fluorescence signalof the labeled cells is then analyzed by flow cytometry, and calculatedas median fluorescence intensity (MFI) of the cell population.

The expression or activity of CXCL16 can be compared to a control.Suitable controls also include a standard value or a cell contacted withthe D ODN in the absence of the agent.

An in vivo method is provided herein for screening for an agent thataffects the immune response induced by a D ODN. In one example, themethod includes administering of the agent to a non-human mammal, suchas a mouse, rat, cat, sheep, dog, goat, pig or monkey, and assessing theexpression and/or activity of CXCL16 in a sample taken from thenon-human mammal.

An increase in the expression and/or activity of CXCL16 indicates thatthe agent is of use in increasing the uptake of a D ODN. An increase inthe expression or activity of CXCL16 also indicates that the agent willincrease an immune response induced by an ODN. A decrease in theexpression and/or activity of CXCL16 indicates that the agent is of usein decreasing the uptake of a D ODN. A decrease in the expression oractivity of CXCL16 also indicates that the agent will decrease an immuneresponse induced by an ODN.

As noted above, the expression or activity of CXCL16 can be measured byany method known to those of skill in the art. For example, theexpression of CXCL16 can be measured by polymerase chain reaction,Western blot, and ELISA and flow cytometry (see Abel et al., J. Immunol.172:6362-6372, 2004, for a detailed description of these methods). Inaddition, an immune response can be evaluated in the animal model. Forexample, the production of a cytokine or the number of cells (such asactivated T cells) can be measured.

The expression or activity of CXCL16 can be compared to a control.Suitable controls also include a standard value. Suitable controls alsoinclude a sample obtained from an animal administered the D ODN in theabsence of the agent.

The disclosure is illustrated by the following non-limiting Examples.

EXAMPLES Example 1 Dextran Sulfate Blocks D ODN Uptake and Signaling

To evaluate whether a scavenger receptor (SR) played a role in D ODNuptake and signaling, the ability of dextran sulfate and fucoidan(classical scavenger receptor ligands) to block the binding of CpG ODNto pDC was evaluated (Table 2 and Supplementary FIG. 3A).

TABLE 2 Effect of dextran sulfate on cellular activation induced by CpGODN Effect of Dextran Sulfate K ODN D ODN % inhibition of binding to23.2 ± 08.8 65.02 ± 5.8  pDC^(a) % inhibition of cytokine 5.8 ± 2.3 90.3± 8.4 secretion^(b) % inhibition in the 0.8 ± 1.8 92.3 ± 5.8 generationof HLA- DR/CD54 double positive cells^(c) ^(a)Purified pDC werepreincubated (20 min at 4° C.) with medium or with 50 μg/ml of dextransulfate followed by addition of 1 μM FITC conjugated ODN. Meanfluorescence intensity representing binding of FITC-ODN was assessed byflow cytometry. ^(b)“K” ODN induced TNF-α production and “D” ODN inducedIFN-α production from PBMC was determined by intracytoplasmic stainingand by ELISA, respectively in the absence or presence of 50 μg/ml ofdextran sulfate. ^(c)Purified pDC were incubated with 1 μM of “K or with3 μM “D” ODN in the absence or presence of 50 μg/ml dextran sulfate andupregulation of HLA-DR/CD54 was determined 24 hours later using flowcytometry. Results represent the mean ± S.D of three independentexperiments.

Dextran sulfate inhibited the binding of D but not K ODN to pDC by >50%(p<0.001). Similar levels of inhibition were achieved in the presence ofanother SR ligand fucoidan but not with the non-ligand chondroitinsulfate. These SR ligands also abolished D ODN induced IFNα secretionand up-regulation of HLA-DR/CD54 by >90% (p<0.001) (Table 2 andSupplementary FIGS. 3B and 3D) but failed to affect K ODN induced TNFαproduction and HLA-DR/CD54 up-regulation (Table 2 and FIGS. 3C and 3D).These results suggested a SR-mediated recognition mechanism for D ODN.

Example 2 Identification of the Receptor for D ODN

To identify the nature of the D ODN receptor, a preliminary screeningfor multiple SRs (type A SR, CD36, CD163, MARCO and SR-PSOX/CXCL16)provided evidence that CXCL16 might be involved in the recognition of DODN. CXCL16 is an unusual chemokine with SR activity when expressed as atransmembrane molecule at the surface of professional antigen presentingcells (Shimaoka et al., J Biol Chem. 275(52):40663-6, 2000). Based onthe initial screening results, the ability of biotin conjugated CpG ODNto bind to purified recombinant CXCL16 was assessed in a cell-freeELISA-based assay and was found to be dose-dependent and significantlyhigher (P<0.001) for D ODN than the binding levels seen with K ODN (FIG.1A).

Example 3 Specificity of CXCL16 for D ODN Binding

Next, the specificity of CXCL16 as a potential binding receptor for DODN was determined in HEK293 cells. The percent of HEK293 cellsinternalizing large amounts of D ODN rose significantly by >5-foldfollowing transfection with a CXCL16 encoding plasmid (p<0.05, FIG. 1B,histogram) as opposed to no change in the case of K ODN. Confocalmicroscopy revealed that CXCL16 colocalized with D but not with K ODN(FIG. 1B). Of note, the level of CXCL16 surface expression correlatedwith the magnitude of D ODN uptake (FIG. 1B, compare cells indicatedwith open versus filled arrow heads).

To evaluate whether CXCL16 could act as a D ODN-specific cell surfacereceptor in pDC, expression of this receptor in human pDC was analyzed.Results from flow cytometry studies showed that 20-40% of pDC (FIG. 4A)expressed CXCL16 on their surface. By comparison, Uchiyama et al.(Tabata S. et al., J Leukoc Biol. 77(5):777-86, 2005) described that pDCproduce CXCL16, but report that most of this material is cleaved andsecreted.

After confirming that pDC express CXCL16 at their surface, ODN bindingto this cell type was assessed in the presence of antibodies againstCXCL16. Anti-CXCL16 antibodies significantly inhibited the binding of DODN to pDC (78.39%, P<0.001, FIG. 1C). Isotype-matched control had noeffect on any class of CpG ODN binding. Of note, the CXCL16 antibodyspecifically inhibited D ODN binding only to pDC but not to the BDCA-2negative CD123+ basophils (FIG. 4B) providing further evidence for thespecificity of this inhibition. In contrast, only 10.7% of K ODN bindingcould be inhibited using anti-CXCL16. These results suggest that CXCL16is involved in the binding/uptake of D but not K ODN.

Example 4 Expression of CXCL16 and D ODN Stimulation

To assess whether pDCs expressing CXCL16 were more responsive to D ODNstimulation, TNFα and IFNα production from CXCL16^(bright) ordim/negative cells was analyzed by intracellular cytokine staining (FIG.2A). K but not D ODN produced significant amounts of TNFα from pDCs and83.21±0.74% of all producers were in the CXCL16 negative/dim population.In contrast, D ODNs induced significant amounts of IFNα and almost allof this cytokine (85.51±0.22%) was produced by the CXCL16^(positive)pDCs. Furthermore, stimulation of highly pure sorted CXCL16^(negative)pDC resulted in up-regulation of HLA-DR/CD86 by K but not by D ODNs,whereas as expected both ODNs showed activity in the sortedCXCL16^(positive+negative) population (FIG. 5). Of interest,UV-irradiated HSV-1 showed a very similar activation pattern as observedwith D ODNs. These results suggest a preferential stimulation ofCXCL16^(positive) pDCs by D but not by K ODNs.

Additional studies confirmed that the immunostimulatory activity of Dclass ODNs required recognition by CXCL16. First, anti-CXCL16 antibodies(Abs) reduced D ODNs dependent IFNα production by 55.9% (p<0.005 whencompared to isotype control, FIG. 2B), but had no significant effect onthe ability of K ODNs to stimulate IP-10 production. Second, anti-CXCL16Abs significantly reduced the ability of D (but not K) ODNs to activatepDC as manifest by the up-regulation of HLA-DR/CD86 expression inelutriated monocytes (FIG. 2C, 68.7% suppression as opposed to 1.4%p.<0.001)

Whereas K ODNs strongly induce NF-KB signaling by TLR9 transfectedHEK293 cells, D ODNs are much less active (Vollmer et al, Eur J Immunol.34(1):251-62, 2004). As seen in FIG. 6, K ODNs increase NF-kB signalingby nearly 4-fold, versus only 1-1.3-fold by two different D ODNs. Yetwhen HEK293 cells that stably express TLR9 were transiently transfectedwith CXCL16, they became highly responsive to D ODN stimulation. Theresponse of CXCL16 transfected TLR9 expressing cells as compared to mocktransfected TLR9 expressing cells is presented in FIG. 2D. CXCL16transfection increases background luciferase activity almost 2-fold forall ODNs irrespective of whether they contain CpG or not except for Dclass, where significantly higher responses were achieved (6-fold,P<0.05, FIG. 2D) in the presence of CXCL16.

Example 5 ADAM-10

CXCL16 expressed on the cell surface is cleaved by the metalloproteinaseADAM-10 (Abel et al., J Immunol. 2004 172(10):6362-72, 2004; Gough etal., J Immunol. 172(6):3678-85, 2004). Previous studies demonstratedthat the amount of the cell surface expressed CXCL16 can be increased bytreatment with the metalloproteinase inhibitor GM6001. Based on theobservation that pDC expressing high levels of CXCL16 were moreresponsive to D ODN stimulation, human PBMC were incubated with GM6001followed by K or D ODNs. Metalloproteinase inhibitor treatment increasedthe expression of CXCL16 (FIG. 7) and D ODN responsiveness (2-fold,p<0.05) of pDCs, but had no effect on cells stimulated with K ODN.

Thus, the scavenger receptor CXCL16 plays a key role in mediating thebinding, uptake and subsequent stimulation mediated by D but not K ODNs.Without being bound by theory, the role of CXCL16 in D ODN uptake andactivity explains previously observed differences in the targeting andinternalization of D ODNs compared to other CpG ODN classes.

As described herein, pDC expressing high levels of that SR were also themost responsive to D ODN activation. Indeed, a significant positivecorrelation was noted between CXCL16 expression and cytokine (IFNα)production by D ODN stimulated pDC. Consistent with this observation,treatment with a metalloproteinase inhibitor that increased cell surfaceexpression of CXCL16 also increased responsiveness to D ODNs. Finally,it was demonstrated that HEK293 cells transfected with a plasmidencoding CXCL16 significantly increased their capacity to bind D (butnot K) ODNs.

HEK293 cells transfected to express TLR9 (the cognate receptor for CpGmotifs) responded to stimulation by K ODNs. This observation suggeststhat K-type CpG ODNs do not require CXCL16 for uptake or activity. Incontrast, HEK293 cells transfected with TLR9 alone did not respond to DODNs, but became responsive when co-transfected with CXCL16. Confocalmicroscopy of these transfected cells showed that D ODNs colocalizedextensively with CXCL16, unlike K ODNs.

The selective binding of D but not K ODNs to CXCL16 could be mediated bythe poly G tail unique to D ODNs. The poly G tail present on all D ODNsallows the formation of G-tetrads, and the resultant generation ofhigher order tertiary structures (Kerkman et al, J Biol Chem.280(9):8086-93, 2005). In this context, it appears that D ODNs, with itsnanoparticle forming ability, is recognized specifically by thescavenger receptor CXCL16 and this highly specific interaction andsubsequent internalization may alter the subcellular distribution ofthis ODN.

Human pDC are known to produce high amounts of IFNα when exposed toenveloped viruses. Since the envelope is derived from the host cellmembranes, phosphatidylserine (PS) could to be present in viralenvelopes. In fact, for extracellular herpes simplex virus (HSV), theviral membrane was shown to contain a 3-fold higher concentration of PScompared to the host nuclear membrane (van Genderen et al., Virology200(2):831-6, 1994). PS was one of the first molecules described asbeing recognized by CXCL16 (Shimaoka et al., J Biol Chem.275(52):40663-6, 2000). Therefore, PS that is expressed on viralenvelopes could be recognized by transmembrane CXCL16 on pDC, therebyfacilitating virus internalization and subsequent IFNα production.

Example 6 Cancer Treatment

Cancer cells, such as mammary cancer cells 11A-1 (Cancer Res, 55:3310-7, 1995), Meth A sarcoma cells (DeLeo et al., J Exp Med, 146:720-34, 1977), or MC-38 (Tan et al., J Natl Cancer Inst (Bethesda), 56:871-3, 1976) are. Meth A is passaged as an ascitic tumor. Cells areharvested, counted, and washed with PBS before use. Four days afterinoculation with 1-2×10⁶ 11A-1, MC-38, or Meth A cells into six-week-oldBALB/c mice (via subcutaneous injection in the left flank), smallorganized tumor nodules are seen on histological section. Tumors aremeasured twice weekly in three dimensions with calipers. Growth curvestruncate when the first mouse in the respective group dies.

Prior studies have demonstrated the ability to generate p53-specificresponses after immunization with modified vaccinia Ankara (MVA)expressing WT murine p53 (MVAp53; Espenschied et al., J Immunol, 170:3401-7, 2003.). Immunized mice develop vigorous p53-specific CTLresponses and are able to reject small, established p53-overexpressingMeth A tumors.

In order to accelerate tumor injection, mice are treated with 15 nmol ofD ODN (or a non-CpG ODN control) by intraperitoneal (i.p.) injection ondays 4, 9, and 14. Groups of mice are administered an agent that inducesthe expression of CXCL16, such as antagonists of ADAM-10, for exampleG1254023X or GM6001 in conjunction with the D ODN (see Table 1 for D ODNsequences). On day 5, the mice are immunized i.p. with 5×10⁷ pfu ofMVAp53, 5×10⁷ pfu of MVApp65, or PBS. The subcutaneous tumors weremeasured twice weekly in three dimensions with calipers.

Although MVAp53 and D ODN each separately result in minimal attenuationof tumor growth, all animals developed progressively lethal tumors. Thecombination of D ODN and MVAp53 immunization results in diminished tumoroutgrowth. The combination of D ODN, MVAp53 and an antagonist of ADAM-10significantly decreases tumor growth and/or results in decreased tumorburden.

It will be apparent that the precise details of the methods orcompositions described may be varied or modified without departing fromthe spirit of the described invention. We claim all such modificationsand variations that fall within the scope and spirit of the claimsbelow.

The invention claimed is:
 1. A pharmaceutical composition comprising: atherapeutically effective amount of a D-type oligodeoxynucleotide,wherein the D oligodeoxynucleotide has a sequence: (SEQ ID NO: 21) 5′X₁X₂X₃ Pu₁ Py₂ CpG Pu₃ Py₄ X₄X₅X₆(W)_(M) (G)_(N)-3′

wherein the central CpG motif is unmethylated, Pu is a purinenucleotide, Py is a pyrimidine nucleotide, X and W are any nucleotide, Mis any integer from 0 to 10, and N is any integer from 4 to 10, andwherein the D oligodeoxynucleotide is 18 to 50 nucleotides in length;and a therapeutically effective amount of an agent that increases theexpression and/or activity of CXCL16 wherein the agent is ametalloproteinase inhibitor that is an antagonist of ADAM-10; in apharmaceutically acceptable carrier.
 2. The pharmaceutical compositionof claim 1, wherein the metalloproteinase inhibitor comprises GW280264X,G1254023X, GM6001, or a pharmaceutically acceptable salt thereof.
 3. Thepharmaceutical composition of claim 1, wherein the metalloproteinaseinhibitor comprises an inhibitor of the formula C₁₈H₃₅NO₂:

an inhibitor of the formula C₁₃H₁₄N₄O₃S₂:

an inhibitor of the formula C₂₁H₂₃N₇O₂S₂:

 or an inhibitor of the formula C₂₁H₁₉NO₄S:


4. The pharmaceutical composition of claim 1, wherein the Doligodeoxynucleotide comprises phosphorothioate bases.
 5. Thepharmaceutical composition of claim 1, wherein Pu₁ Py₂ and Pu₃ Py₄ areself-complementary.
 6. The pharmaceutical composition of claim 1,wherein X₁X₂X₃ Pu₁ Py₂ and Pu₃ Py₄ X₄X₅X₆ are self-complementary.
 7. Thepharmaceutical composition of claim 1, wherein the oligodeoxynucleotidecomprises the nucleotide sequence set forth as one of SEQ ID NO: 1, SEQID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ IDNO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ IDNO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16 orSEQ ID NO:
 17. 8. The pharmaceutical composition of claim 1, wherein theoligodeoxynucleotide consists of the nucleotide sequence set forth asone of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ IDNO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ IDNO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQID NO: 15, SEQ ID NO: 16 or SEQ ID NO:
 17. 9. The pharmaceuticalcomposition of claim 1, further comprising a therapeutically effectiveamount of an anti-infectious agent.
 10. The pharmaceutical compositionof claim 9, wherein the anti-infectious agent is an antiviral,antifungal, or anti-bacterial agent.
 11. The pharmaceutical compositionof claim 1, further comprising a vaccine.
 12. The pharmaceuticalcomposition of claim 11, wherein the vaccine is a heat-killed,attenuated or a live vaccine.
 13. The pharmaceutical composition ofclaim 1, further comprising a second agent.
 14. The pharmaceuticalcomposition of claim 1, formulated for oral administration.
 15. Thepharmaceutical composition of claim 1, formulated for topicaladministration.
 16. A pharmaceutical composition comprising atherapeutically effective amount of a D-type oligodeoxynucleotide,wherein the D oligodeoxynucleotide has the nucleotide sequence set forthas SEQ ID NO: 1, and a therapeutically effective amount of GM6001, in apharmaceutically acceptable carrier.
 17. The pharmaceutical compositionof claim 16, further comprising a vaccine.